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A rice thousand-grain weight gene tgw6 mutant and its preparation method and application

A thousand-grain weight, mutant technology, applied in the field of plant biology, can solve the problem of lack of relevant mutant breeding value evaluation and other problems

Active Publication Date: 2018-10-30
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the CRISPR / Cas9 system has successfully achieved targeted genome editing in plants such as Arabidopsis thaliana, tobacco, sweet orange, rice, wheat, sorghum, corn, and bryophytes, but it is important for the yield and quality of rice breeding. There are few reports on the targeted editing of key genes such as , fertility, etc., and there is even a lack of evaluation of the breeding value of related mutants

Method used

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  • A rice thousand-grain weight gene tgw6 mutant and its preparation method and application
  • A rice thousand-grain weight gene tgw6 mutant and its preparation method and application
  • A rice thousand-grain weight gene tgw6 mutant and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0054] Example 1 Creation of rice thousand-grain weight gene tgw6 deletion mutant based on CRISPR / Cas9 technology

[0055](1) Design of guide RNA (guide RNA, gRNA) target sequence According to the genome sequence of the rice grain length and thousand-grain weight gene TGW6 (GenBank: AB513135.1), three gRNAs targeting the thousand-grain weight gene TGW6 were designed. The 20nt oligonucleotide gRNA target sequence is designed according to the A / G(N)20NGG sequence, and the designed gRNA target sequence is compared with the rice genome database to exclude non-specific target cutting sites. The nucleotide sequence is shown in Table 1 and figure 1 .

[0056] Table 1 Oligonucleotide sequences of gRNA targets

[0057]

[0058] (2) Construction of three-target CRISPR / Cas9-gRNA vector

[0059] Refer to Ma et al. (Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z. 2015. Arobust CRISPR / Cas9system for convenient, high-efficiency multiplex genome editing in monocot and dicotp...

Embodiment 2

[0072] (1) Agrobacterium-mediated genetic transformation of rice callus

[0073] The CRISPR / Cas9-gRNA vector assembled in Example 1 was transformed into Agrobacterium EHA105 by electric shock. PCR detection (primers: hpt F: 5'-TCCGGAGCCTCCGCTCGAAGTAG-3', hpt R: 5'-CTGAACTCACCGCGACGTCTGTC-3') positive clones were used to infect rice material H447 (BC of R819 / Yuzhenxiang / / R819 2 f 7 ), the infection method was carried out with reference to the method of Hiei et al. (1994), and transgenic rice plants were obtained.

[0074] (2) Extraction of rice genomic DNA and PCR detection and sequencing analysis of mutants

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Abstract

The invention belongs to the field of plant biotechnology, and in particular relates to a rice thousand kernel weight gene tgw6 mutant as well as a preparation method and application thereof. According to the invention, by designing a specific TGW6 site and by site-directed editing a TGW6 gene for regulating and controlling rice thousand kernel weight by virtue of CRISPR / Cas9 technology, a set of rice tgw6-deleted mutant new germplasm, namely Cas-tgw6-a, Cas-tgw6b or Cas-tgw6c, having an important application value can be obtained, and the mutant can cause significant influence to the thousand kernel weight of rice and can improve rice thousand kernel weight by more than 5%. The tgw6 mutant is applicable to rice high-yield and stable-yield breeding.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, and in particular relates to a rice thousand-grain weight gene tgw6 mutant and a preparation method and application thereof. Background technique [0002] Rice (Oryza sativa L.) is one of the most important food crops in the world, feeding more than half of the world's population and the main source of food for nearly half of my country's population. With the continuous growth of population, people's demand for food is also increasing. The effective use of heterosis has played an important role in promoting the increase of rice yield. However, the rice yield per unit area has not been greatly improved, and with the reduction of cultivated land, the planting area of ​​rice has been declining. It is urgent to use new Genetic improvement strategies to greatly increase rice yield. In recent years, with several genes controlling grain size (GS3, SW5 and GW8), grain width and grain weight (GW2, GW5...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82
Inventor 王加峰黄翠红郭涛罗文龙陈志强王慧
Owner SOUTH CHINA AGRI UNIV
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